Experimental analysis of second cleavage in the mouse

BACKGROUND: Mammalian conceptuses typically have an approximately regular tetrahedral shape at the 4-cell stage. In the rabbit, this has been attributed to both 2-cell blastomeres dividing meridionally, but with the animal-vegetal axis of the second blastomere to divide rotating through roughly 90 degrees before or during cytokinesis. The aim of the present study was to ascertain whether this was also true for the mouse. METHODS AND RESULTS: First, the distribution in regular tetrahedral 4-cell conceptuses of fluorescent microspheres applied to the vegetal polar region of one or both blastomeres at the 2-cell stage was analysed. Second, the ability of 2-cell stages to form regular tetrahedral 4-cell conceptuses after the previtelline space had been gelated to prevent blastomeres from rotating was also investigated. Neither experiment yielded evidence supporting blastomere rotation during second cleavage. Rather, the findings were consistent with the regular tetrahedral form of 4-cell conceptus resulting from meridional division of one blastomere and approximately equatorial division of the other. CONCLUSIONS: Second cleavage in the mouse typically yields 4-cell conceptuses with three distinct types of blastomere. While both products of the meridional division include all axial levels of the zygote, those of the equatorial division acquire only its vegetal or animal half.     

Lineage and fate of each blastomere of the eight-cell sea urchin embryo

A fluoresceinated lineage tracer was injected into individual blastomeres of eight-cell sea urchin (Strongylocentrotus purpuratus) embryos, and the location of the progeny of each blastomere was determined in the fully developed pluteus. Each blastomere gives rise to a unique portion of the advanced embryo. We confirm many of the classical assignments of cell fate along the animal-vegetal axis of the cleavage-stage embryo, and demonstrate that one blastomere of the animal quartet at the eight-cell stage lies nearest the future oral pole and the opposite one nearest the future aboral pole of the embryo. Clones of cells deriving from ectodermal founder cells always remain contiguous, while clones of cells descendant from the vegetal plate (i.e., gut, secondary mesenchyme) do not. The locations of ectodermal clones contributed by specific blastomeres require that the larval plane of bilateral symmetry lie approximately equidistant (i.e., at a 45 degree angle) from each of the first two cleavage planes. These results underscore the conclusion that many of the early spatial patterns of differential gene expression observed at the molecular level are specified in a clonal manner early in embryonic sea urchin development, and are each confined to cell lineages established during cleavage.     

Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres

Summary A detailed fate map of all the progeny derived from each of the blastomeres of the 4- and 8-cell stage South African clawed frog (Xenopus laevis) embryo is presented. Each “identified” blastomere that results from stereotypic cleavages has a characteristic set of progeny that distinguishes it from the other blastomeres of the embryo. The 4-cell dorsal (D) blastomere is the major progenitor of the stomodeum, cement gland, retina, notochord, head somite, pharynx and liver. The 4-cell ventral (V) blastomere is the major progenitor of the trunk and fin epidermis, ventral somite, nephrotome, lateral plate mesoderm and proctodeum. The other organs are derived from both blastomeres. At the next cell division, the animal hemisphere daughters of both blastomeres (D1 and V1, respectively) become the major progenitors for head ectodermal and mesodermal structures, and the vegetal hemisphere daughters become the major progenitors for trunk mesodermal (D2) or trunk endodermal (V2) structures. Semiquantitative lineage diagrams, using data from this and from previous studies demonstrate that as cleavage proceeds from the 2- to the 32-cell stage, the progenitors for particular organs or for specific regions of organs segregate into defined regions of the blastula. To determine whether this segregation is related to the position of the blastomere or to its geneological lineage, we compared the fates of radial 8-cell blastomeres to those of stereotypic 8-cell blastomeres. Radial blastomeres have fates nearly equivalent to the sum of the two 16-cell blastomeres that occupy the same position in the embryo, demonstrating that fate depends upon blastomere position rather than lineage.     

Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite

Embryonic patterning in vertebrates is dependent upon the balance of inductive signals and their specific antagonists. We show that Noggin, which encodes a bone morphogenetic protein (BMP) antagonist expressed in the node, notochord, and dorsal somite, is required for normal mouse development. Although Noggin has been implicated in neural induction, examination of null mutants in the mouse indicates that Noggin is not essential for this process. However, Noggin is required for subsequent growth and patterning of the neural tube. Early BMP-dependent dorsal cell fates, the roof plate and neural crest, form in the absence of Noggin. However, there is a progressive loss of early, Sonic hedgehog (Shh)-dependent ventral cell fates despite the normal expression of Shh in the notochord. Further, somite differentiation is deficient in both muscle and sclerotomal precursors. Addition of BMP2 or BMP4 to paraxial mesoderm explants blocks Shh-mediated induction of Pax-1, a sclerotomal marker, whereas addition of Noggin is sufficient to induce Pax-1. Noggin and Shh induce Pax-1 synergistically. Use of protein kinase A stimulators blocks Shh-mediated induction of Pax-1, but not induction by Noggin, suggesting that induction is mediated by different pathways. Together these data demonstrate that inhibition of BMP signaling by axially secreted Noggin is an important requirement for normal patterning of the vertebrate neural tube and somite.     

Computer-controlled, multilevel, morphometric analysis of blastomere size as biomarker of fragmentation and multinuclearity in human embryos

BACKGROUND: Little is known about blastomere size at different cleavage stages and its correlation with embryo quality in human embryos. Using a computer system for multilevel embryo morphology analysis we have analysed blastomeres of human embryos and correlated mean blastomere size with embryonic fragmentation and multinuclearity. METHODS: A consecutive cohort of 232 human 2-, 3- and 4-cell embryos from patients referred for ICSI treatment were included. Sequences of digital images were taken by focusing at 5- micro m intervals through the embryo. Blastomere sizes and number of nuclear structures were evaluated based on these sequences. The degree of embryonic fragmentation was evaluated by normal morphological assessment prior to transfer and correlated to the blastomere sizes. RESULTS: As a result of normal cell cleavage, mean blastomere size decreased significantly from a volume of 0.28 x 10(6) microm(3) at the 2-cell stage to 0.15 x 10(6) microm(3) at the 4-cell stage (P < 0.001). Mean blastomere size decreased significantly (P < 0.001) with increasing degree of embryonic fragmentation, where highly fragmented embryos showed a 43-67% reduction in blastomere volume compared with embryos with no fragmentation. Multinucleated blastomeres were significantly larger than non-multinucleated blastomeres (P < 0.001). On average, multinucleated blastomeres were 51.5, 67.8 and 73.1% larger than their non-multinucleated sibling blastomeres at the 2-, 3- and 4-cell stage, respectively. Furthermore, the average volume of non-multinucleated blastomeres originating from multinucleated embryos was significantly smaller than the average volume of the blastomeres from mononucleated embryos (P < 0.001). CONCLUSIONS: The results of this study show that the average blastomere size is significantly affected by degree of fragmentation and multinuclearity, and that computer-assisted, multilevel analysis of blastomere size may function as a biomarker for embryo quality.     

Endogenous bone morphogenetic protein antagonists regulate mammalian neural crest generation and survival.

We demonstrate here that Chordin and Noggin function as bone morphogenetic protein (BMP) antagonists in vivo to promote mammalian neural crest development. Using Chrd and Nog single and compound mutants, we find that Noggin has a major role in promoting neural crest formation, in which Chordin is partially redundant. BMP signaling is increased in dorsal tissues lacking Noggin and is further increased when Chordin is also absent. The early neural crest domain is expanded with decreased BMP antagonism in vivo. Noggin and Chordin also regulate subsequent neural crest cell emigration from the neural tube. However, reduced levels of these BMP antagonists ultimately result in perturbation of neural crest cell derived peripheral nervous system and craniofacial skeletal elements. Such defects reflect, at least in part, a function to limit apoptosis in neural crest cells. Noggin and Chordin, therefore, function together to regulate both the generation and survival of neural crest cells in mammalian development.     

PCR技术用于单个卵裂球基因诊断的实验研究

目的建立单卵裂球PCR技术,为开展单基因病的着床前遗传学诊断奠定基础。方法共对103个卵裂球进行了PCR扩增;其中30个扩增KG8位点,33个扩增D16S291位点,40个扩增D16S423位点。结果单卵裂球扩增KG8、D16S291和D16S423的成功率分别86.7%、87.9%和87.5%。结论建立的单卵裂球PCR技术是稳定可靠的,可以用于单基因病的着床前遗传学诊断。     

Antagonists of Wnt and BMP signaling promote the formation of vertebrate head muscle

Recent studies have postulated that distinct regulatory cascades control myogenic differentiation in the head and the trunk. However, although the tissues and signaling molecules that induce skeletal myogenesis in the trunk have been identified, the source of the signals that trigger skeletal muscle formation in the head remain obscure. Here we show that although myogenesis in the trunk paraxial mesoderm is induced by Wnt signals from the dorsal neural tube, myogenesis in the cranial paraxial mesoderm is blocked by these same signals. In addition, BMP family members that are expressed in both the dorsal neural tube and surface ectoderm are also potent inhibitors of myogenesis in the cranial paraxial mesoderm. We provide evidence suggesting that skeletal myogenesis in the head is induced by the BMP inhibitors, Noggin and Gremlin, and the Wnt inhibitor, Frzb. These molecules are secreted by both cranial neural crest cells and by other tissues surrounding the cranial muscle anlagen. Our findings demonstrate that head muscle formation is locally repressed by Wnt and BMP signals and induced by antagonists of these signaling pathways secreted by adjacent tissues.     

Regulation of dorsal somitic cell fates: BMPs and Noggin control the timing and pattern of myogenic regulator expression

Previous work has indicated that signals from the neural tube, notochord, and surface ectoderm promote somitic myogenesis. Here, we show that somitic myogenesis is under negative regulation as well; BMP signaling serves to inhibit the activation of MyoD and Myf5 in Pax3-expressing cells. Furthermore, we show that the BMP antagonist Noggin is expressed within the dorsomedial lip of the dermomyotome, where Pax3-expressing cells first initiate the expression of MyoD and Myf5 to give rise to myotomal cells in the medial somite. Consistent with the expression of Noggin in dorsomedial dermomyotomal cells that lie adjacent to the dorsal neural tube, we have found that coculture of somites with fibroblasts programmed to secrete Wnt1, which is expressed in dorsal neural tube, can induce somitic Noggin expression. Ectopic expression of Noggin lateral to the somite dramatically expands MyoD expression into the lateral regions of the somite, represses Pax3 expression in this tissue, and induces formation of a lateral myotome. Together, our findings indicate that the timing and location of myogenesis within the somite is controlled by relative levels of BMP activity and localized expression of a BMP antagonist.     

Sinistral equal-size spiral cleavage of the indirectly developing polychaete Hydroides elegans.

Two major variants of the stereotypic spiral cleavage correlate with distinct developmental modes in polychaetes. Indirect development through a feeding trochophore larva correlates with development from four equal-sized blastomeres, whereas direct development correlates with unequal cleavage characterized by a large dorsal blastomere precursor maternally predetermined. The equal-size spiral cleavage of the indirectly developing serpulid Hydroides elegans has been reconstructed from serial sections of nuclei-stained embryos. The order of cell divisions has been determined from the 2-cell stage to the 80-cell stage, when gastrulation cell movements start to overlap with late spiral-cleavage divisions. In contrast to related species, the third cleavage in Hydroides elegans is invariably sinistral. The four quadrants remain indistinct until the 60-cell stage, when the small 2d22 and large 2d21 cells are generated. The developmental significance of the invariant spiral cleavage relates to the spatial distribution of gene functions that it partitions and their relation to blastomere fate commitments. The conservation and divergence of the cleavage pattern among spiralians is well suited to study the developmental control of the cell-cleavage machinery and its evolution.     

Planar induction of convergence and extension of the neural plate by the organizer of Xenopus.

This paper demonstrates that convergence and extension within the neural plate of Xenopus laevis are regulated by planar inductive interactions with the adjacent Spemann organizer. The companion article (Keller et al.: Developmental Dynamics 193:199-217, 1992) showed that the prospective hindbrain and spinal cord occupy a very short and very wide area just above the Spemann organizer in the early gastrula and that these regions converge and extend greatly during gastrulation and neurulation, using a sequence of radial and mediolateral cell intercalations. In this article, we show that "planar" contact of these regions with the organizer at their vegetal edge until stage 11 is sufficient to induce convergence and extension, after which their convergence and extension become autonomous. Grafts of the organizer in planar contact with uninduced ectodermal tissues induce these ectodermal tissues to converge and extend by a planar inductive signal from the organizer. Labeling of the inducing or responding tissues confirms that only planar interactions occur. Neural convergence and extension are actually hindered in explants deliberately constructed so that vertical interactions occur. These results show unambiguously that the Spemann organizer induces the extraordinary and precocious convergence and extension movements of the Xenopus neural plate by planar interactions acting over short distances.     

BMP signaling regulates the fate of chondro‐osteoprogenitor cells in facial mesenchyme in a stage‐specific manner

Background : Lineage tracing has shown that most of the facial skeleton is derived from cranial neural crest cells. However, the local signals that influence postmigratory, neural crest‐derived mesenchyme also play a major role in patterning the skeleton. Here, we study the role of BMP signaling in regulating the fate of chondro‐osteoprogenitor cells in the face. Results : A single Noggin‐soaked bead inserted into stage 15 chicken embryos induced an ectopic cartilage resembling the interorbital septum within the palate and other midline structures. In contrast, the same treatment in stage 20 embryos caused a loss of bones. The molecular basis for the stage‐specific response to Noggin lay in the simultaneous up‐regulation of SOX9 and downregulation of RUNX2 in the maxillary mesenchyme, increased cell adhesiveness as shown by N‐cadherin induction around the beads and increased RA pathway gene expression. None of these changes were observed in stage 20 embryos. Conclusions : These experiments demonstrate how slight changes in expression of growth factors such as BMPs could lead to gain or loss of cartilage in the upper jaw during vertebrate evolution. In addition, BMPs have at least two roles: one in patterning the skull and another in regulating the skeletogenic fates of neural crest‐derived mesenchyme. Developmental Dynamics 245:947–962, 2016. © 2016 Wiley Periodicals, Inc.     

Rab3d is required for Xenopus anterior neurulation by regulating noggin secretion

Rab3d is a member of the Ras‐related small GTPase family of secretory Rab, Rab3. In this study, we showed that Xenopus Rab3d is expressed specifically in the anterior border of the neural plate when the neural plate converges and folds to initiate neural tube formation. Morpholino‐mediated knockdown of Rab3d resulted in neurulation defects both in neural plate convergence and folding. Interestingly, perturbation of BMP signaling rescued neurulation defects of Rab3d morphants, suggesting that Rab3d inhibits BMP signaling during neurulation. By secretion assay in the Xenopus animal cap, we found that Rab3d specifically regulates secretion of a BMP antagonist, Noggin, but not Chordin and Wnts. We also found that Rab3d is co‐localized with Noggin and that this interaction is dependent on the GTP/GDP cycle of Rab3d. Collectively, these findings suggest that Rab3d‐mediated secretion regulation of a BMP antagonist, Noggin, is one of the mechanisms of BMP antagonism during Xenopus anterior neurulation. Developmental Dynamics 240:1430–1439, 2011. © 2011 Wiley‐Liss, Inc.     

Bone morphogenetic protein signaling is required in the dorsal neural folds before neurulation for the induction of spinal neural crest cells and dorsal neurons

Bone Morphogenetic Protein (BMP) activity has been implicated as a key regulator of multiple aspects of dorsal neural tube development. BMP signaling in the dorsal‐most neuroepithelial cells presumably plays a critical role. We use tissue‐specific gene ablation to probe the roles of BMPR1A, the type 1 BMP receptor that is seemingly the best candidate to mediate the activities of BMPs on early dorsal neural development. We use two different Cre lines expressed in the dorsal neural folds, one prior to spinal neurulation and one shortly afterward, together with a Bmpr1a conditional null mutation. Our findings indicate that BMPR1A signaling in the dorsal neural folds is important for hindbrain neural tube closure, but suggest it is dispensable for spinal neurulation. Our results also demonstrate a requirement for BMP signaling in patterning of dorsal neural tube cell fate and in neural crest cell formation, and imply a critical period shortly before neural tube closure. Developmental Dynamics 240:755–765, 2011. © 2011 Wiley‐Liss, Inc.     

Multiple maternal influences on dorsal-ventral fate of Xenopus animal blastomeres.

Molecular asymmetries in the animal-vegetal axis of the Xenopus oocyte are well known to regulate the formation of gametes and germ layers. Likewise, many transplantation and explant studies demonstrate that maternal dorsalizing activities are localized to the future dorsal side of the embryo after fertilization, but to date only a few of the molecules involved in this process have been shown to be asymmetrically distributed. In this report, we identify two new aspects of the maternal regulation of dorsal-ventral fate asymmetry in Xenopus blastomeres: cytoplasmic polyadenylation of dorsal maternal mRNAs and localized Wnt8b signaling. Previous studies demonstrated that there are maternal, dorsal axis-inducing RNAs localized to dorsal animal blastomeres that become activated between the 8- and 16-cell stage (Hainski and Moody [1992] Development 116:347-355; Hainski and Moody [1996] Dev. Genet. 19:210-221). We report herein that the activation of these axis-inducing dorsal mRNAs is regulated by cytoplasmic polyadenylation. We also show that maternal wnt8b mRNA is concentrated in ventral animal blastomeres. These ventral cells and exogenous Wnt8b both inhibit the dorsal fate of neighboring blastomeres in culture, indicating that a maternal Wnt signal also contributes to segregating dorsal and ventral fates.     

Zona pellucida damage to human embryos after cryopreservation and the consequences for their blastomere survival and in-vitro viability

The study objective was to quantify zona pellucida (ZP) damage in cryopreserved human embryos. The influence of two different freezing containers was investigated, and the influence of freezing damage on the survival and viability of the embryos evaluated. ZP damage did not differ according to whether embryos originated from in-vitro fertilization (IVF) cycles or from IVF cycles in association with intracytoplasmic sperm injection (ICSI). The freezing container, however, significantly influenced the occurrence of ZP damage after cryopreservation. More damage was observed when the embryos were frozen-thawed using plastic cryovials than using plastic mini-straws (16.6% versus 2.3%; P < 0.0001). A clear association was found between blastomere survival and ZP intactness. Consequently, the percentage of embryos with 100% blastomere survival was higher when embryos were frozen-thawed using plastic mini-straws. The further cleavage of frozen-thawed embryos suitable for transfer was not different whether there was ZP damage or not; however, it was higher when there was 100% blastomere survival as compared with when some blastomeres were damaged (79.0% versus 43.7%; P < 0.0001). Consequently, more embryos suitable for transfer cleaved further when they were frozen-thawed using plastic mini-straws. In conclusion, the aim of a cryopreservation programme should be to have as many fully intact embryos as possible after thawing. Increased ZP damage might indicate a suboptimal cryopreservation procedure.     

Noggin null allele mice exhibit a microform of holoprosencephaly

Holoprosencephaly (HPE) is a heterogeneous craniofacial and neural developmental anomaly characterized in its most severe form by the failure of the forebrain to divide. In humans, HPE is associated with disruption of Sonic hedgehog and Nodal signaling pathways, but the role of other signaling pathways has not yet been determined. In this study, we analyzed mice which, due to the lack of the Bmp antagonist Noggin, exhibit elevated Bmp signaling. Noggin(-/-) mice exhibited a solitary median maxillary incisor that developed from a single dental placode, early midfacial narrowing as well as abnormalities in the developing hyoid bone, pituitary gland and vomeronasal organ. In Noggin(-/-) mice, the expression domains of Shh, as well as the Shh target genes Ptch1 and Gli1, were reduced in the frontonasal region at key stages of early facial development. Using E10.5 facial cultures, we show that excessive BMP4 results in reduced Fgf8 and Ptch1 expression. These data suggest that increased Bmp signaling in Noggin(-/-) mice results in downregulation of the hedgehog pathway at a critical stage when the midline craniofacial structures are developing, which leads to a phenotype consistent with a microform of HPE.     

Identification of neural genes using Xenopus DNA microarrays.

To isolate novel genes regulating neural induction, we used a DNA microarray approach. As neural induction is thought to occur by means of the inhibition of bone morphogenetic protein (BMP) signaling, BMP signaling was inhibited in ectodermal cells by overexpression of a dominant-negative receptor. RNAs were isolated from control animal cap explants and from dominant-negative BMP receptor expressing animal caps and subjected to a microarray experiment using newly generated high-density Xenopus DNA microarray chips representing over 17,000 unigenes. We have identified 77 genes that are induced in animal caps after inhibition of BMP signaling, and all of these genes were subjected to whole-mount in situ hybridization analysis. Thirty-two genes showed specific expression in neural tissues. Of the 32, 14 genes have never been linked to neural induction. Two genes that are highly induced by BMP inhibition are inhibitors of Wnt signaling, suggesting that a key step in neural induction is to produce Wnt antagonists to promote anterior neural plate development. Our current analysis also proves that a microarray approach is useful in identifying novel candidate factors involved in neural induction and patterning.